This disclosure describes compositions and methods for enhanced production of enduracidin in genetically engineered strains of Streptomyces fungicidicus. In particular, the present disclosure describes the genetic manipulation of regulatory genes orf24 and orf18 associated with the enduracidin (enramycin) biosynthesis gene cluster from Streptomyces fungicidicus to generate vector constructs and recombinant strains producing greater yields of enduracidin.

Disclosed is a high-throughput transcriptional assay format in Actinomycete bacteria, and Streptomyces spp. in particular, that leverages eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, a silent gene cluster in Streptomyces albus J1074 was induced. The cytotoxins etoposide and ivermectin were revealed as potent inducers, allowing the isolation and structural characterization of nearly 20 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under normal growth conditions. The successful implementation of this approach will allow future discovery of cryptic metabolites with useful bioactivities from Actinomycete bacteria.

Disclosed is a high-throughput transcriptional assay format in Actinomycete bacteria, and Streptomyces spp. in particular, that leverages eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, a silent gene cluster in Streptomyces albus J1074 was induced. The cytotoxins etoposide and ivermectin were revealed as potent inducers, allowing the isolation and structural characterization of nearly 20 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under normal growth conditions. The successful implementation of this approach will allow future discovery of cryptic metabolites with useful bioactivities from Actinomycete bacteria.

The present disclosure provides a spiramycin-producing strain, a carrimycin-producing strain, construction method therefor, use thereof and method for increasing the product yield thereof. The provided spiramycin-producing strain has an inactivated gene Lrp (Δlrp-SP); and the strain has a preservation number of CGMCC No.16056. The provided carrimycin-producing strain has an inactivated gene Lrp (Δlrp-BT); and the strain has a preservation number of CGMCC No.16055. By inactivating the gene Lrp, the yields of spiramycin and carrimycin are increased; and particularly the yield and proportion of a major component of the carrimycin, that is, 4″-O-isovalerylspiramycin III are significantly increased.

The present disclosure provides a spiramycin-producing strain, a carrimycin-producing strain, construction method therefor, use thereof and method for increasing the product yield thereof. The provided spiramycin-producing strain has an inactivated gene Lrp (Δlrp-SP); and the strain has a preservation number of CGMCC No.16056. The provided carrimycin-producing strain has an inactivated gene Lrp (Δlrp-BT); and the strain has a preservation number of CGMCC No.16055. By inactivating the gene Lrp, the yields of spiramycin and carrimycin are increased; and particularly the yield and proportion of a major component of the carrimycin, that is, 4″-O-isovalerylspiramycin III are significantly increased.

Provided are biologically pure bacterial isolates characterized by a genome structure at least 90% similar to a genome structure of a bacterial species selected from the group consisting of: Streptomyces sp. E128 having an NRRL Accession No. B-67462, Bacillus amyloliquefaciens A190 having an NRRL Accession No. B-67464, Bacillus subtilis P243 having an NRRL Accession No. B-67459, Bacillus thuringiensis M979 having an NRRL Accession No. B-67457, Massilia aurea P63 having an NRRL Accession No. B-67461, Rhodococcus sp. G706, Stenotrophomonas maltophilia E132 having an NRRL Accession No. B-67460, Streptomyces aurantiacus A918, Streptomyces badius 0180, Streptomyces mirabilis B670 having an NRRL Accession No. B67463, Streptomyces scopuliridis F427 having an NRRL Accession No. B-67458, and Streptomyces sp. L219. Also provided are whole cell broth or lysates thereof, and polynucleotide, polypeptides and constructs expressing same, compositions-of-matter comprising same and methods using same for killing or inhibiting the development of insects.

Provided are biologically pure bacterial isolates characterized by a genome structure at least 90% similar to a genome structure of a bacterial species selected from the group consisting of: Streptomyces sp. E128 having an NRRL Accession No. B-67462, Bacillus amyloliquefaciens A190 having an NRRL Accession No. B-67464, Bacillus subtilis P243 having an NRRL Accession No. B-67459, Bacillus thuringiensis M979 having an NRRL Accession No. B-67457, Massilia aurea P63 having an NRRL Accession No. B-67461, Rhodococcus sp. G706, Stenotrophomonas maltophilia E132 having an NRRL Accession No. B-67460, Streptomyces aurantiacus A918, Streptomyces badius 0180, Streptomyces mirabilis B670 having an NRRL Accession No. B67463, Streptomyces scopuliridis F427 having an NRRL Accession No. B-67458, and Streptomyces sp. L219. Also provided are whole cell broth or lysates thereof, and polynucleotide, polypeptides and constructs expressing same, compositions-of-matter comprising same and methods using same for killing or inhibiting the development of insects.

Provided are biologically pure bacterial isolates characterized by a genome structure at least 90% similar to a genome structure of a bacterial species selected from the group consisting of: Streptomyces sp. E128 having an NRRL Accession No. B-67462, Bacillus amyloliquefaciens A190 having an NRRL Accession No. B-67464, Bacillus subtilis P243 having an NRRL Accession No. B-67459, Bacillus thuringiensis M979 having an NRRL Accession No. B-67457, Massilia aurea P63 having an NRRL Accession No. B-67461, Rhodococcus sp. G706, Stenotrophomonas maltophilia E132 having an NRRL Accession No. B-67460, Streptomyces aurantiacus A918, Streptomyces badius O180, Streptomyces mirabilis B670 having an NRRL Accession No. B67463, Streptomyces scopuliridis F427 having an NRRL Accession No. B-67458, and Streptomyces sp. L219. Also provided are whole cell broth or lysates thereof, and polynucleotide, polypeptides and constructs expressing same, compositions-of-matter comprising same and methods using same for killing or inhibiting the development of insects.

Provided are biologically pure bacterial isolates characterized by a genome structure at least 90% similar to a genome structure of a bacterial species selected from the group consisting of: Streptomyces sp. E128 having an NRRL Accession No. B-67462, Bacillus amyloliquefaciens A190 having an NRRL Accession No. B-67464, Bacillus subtilis P243 having an NRRL Accession No. B-67459, Bacillus thuringiensis M979 having an NRRL Accession No. B-67457, Massilia aurea P63 having an NRRL Accession No. B-67461, Rhodococcus sp. G706, Stenotrophomonas maltophilia E132 having an NRRL Accession No. B-67460, Streptomyces aurantiacus A918, Streptomyces badius O180, Streptomyces mirabilis B670 having an NRRL Accession No. B67463, Streptomyces scopuliridis F427 having an NRRL Accession No. B-67458, and Streptomyces sp. L219. Also provided are whole cell broth or lysates thereof, and polynucleotide, polypeptides and constructs expressing same, compositions-of-matter comprising same and methods using same for killing or inhibiting the development of insects.

The present disclosure includes genetically engineered, non-pathogenic Streptomyces bacterium with exogenous, non-native Thaxtomin A (ThxA) biosynthetic gene clusters conferring the genetically engineered, non-pathogenic Streptomyces bacterium with the ability to produce thaxtomin A. Also included are methods of providing thaxtomin producing capability in non-native Streptomyces bacterial strains, methods of producing thaxtomin compounds with the genetically engineered Streptomyces bacteria of the present disclosure, and methods of producing thaxtomin compounds and nitro-tryptophan analogs, and fluorinated thaxtomin compounds, analogs, and intermediates with the genetically engineered Streptomyces bacteria of the present disclosure.